The invention relates to a device for purifying nucleic acids from a formalin-fixed and paraffin-embedded sample comprising a hollow body having a sample inlet port and an outlet port and a filter layer arranged within the hollow body.
In the context of the histopathological examinations of biopsy material, formalin fixation with subsequent paraffin embedding is a procedure that has been known for many years. Morphological changes in such samples are largely excluded. This technique therefore presents an opportunity for permanent preservation of tissue samples. This method is also used in the medical field in the context of cancer disease, where a purely morphological characterization of the tissue samples is often insufficient to identify an ideal treatment method. In this context, a genetic analysis of the material is becoming increasingly important.
A fixed sample is presently understood to mean a biological sample, which is preserved by methods known per se, for example, using a formaldehyde solution. To further improve stability during storage, these samples are then placed in paraffin. Samples treated with formalin and embedded in paraffin are also referred to as FFPE tissues (“formalin-fixed, paraffin-embedded”). The sample fixed and embedded by this method can be used, for example, for histopathological examinations and/or subsequently stored over a very long period without any appreciable changes occurring in the biomolecules contained in these samples. In particular, even after longer periods, nucleic acids, i.e. RNA and DNA, can still be extracted from these samples.
The extraction of these biomolecules from such fixed, paraffin-embedded biological samples is, however, laborious because the samples must first be liberated from the paraffin they are surrounded by. The samples are usually thin sections in which the paraffin portion is generally in significant excess of the sample portion. Because the paraffin can interfere with, or completely prevent, further sample preparation and isolation of the biomolecules, different preparation methods are proposed in the state of the art by means of which the paraffin can be separated.
In light of the currently already very large and ever-expanding FFPE-libraries, methods that allow manual extraction on a small-scale only are hardly suitable to efficiently utilize the libraries in retrospective studies. In order to avoid a selection bias of the results as far as possible, it is necessary in this context to develop a method, which is completely automated to the highest extent possible and thereby reduces manual labor time to a minimum. Only in this way can sample throughput be substantially increased.
For throughput of large numbers of samples, however, the methods known to date, which can be performed only with large amounts of hazardous substances, are not suitable.
In general, for sample analysis with molecular biology methods, a small portion of the total embedded biopsy material must be treated in a special way. This involves first removing the paraffin and dissolving the samples in order to release the nucleic acids.
De-paraffinization of biological samples is known, for example, from EP 1 242 594. Paraffin is removed by repeated washing of the paraffin-embedded samples first with organic solvents such as, for example, xylene. Then, alcohol is used to remove the solvent and rehydrate the tissue. To this end, the sample is washed several times in succession, centrifuged, and the wash solutions lastly removed from the reaction tube by pipetting. In conventional automated liquid-handling pipetting devices, centrifugation steps can be implemented only with considerable effort and with the use of special automation-adapted centrifuges. In addition, pipette tips can become clogged by sample- and/or tissue residues during removal of the wash solutions by pipetting. This can lead to uncontrolled and incomplete liquid removal. Potentially remaining alcohol from the final wash steps can further interfere with subsequent enzymatic sample lysis.
Other methods are known from the state of the art that do not require aromatic solvents, such as xylene. Such procedures are described, for example, in Bohmann K et al 2009: RNA Extraction from Archival Formalin-Fixed Paraffin-Embedded Tissue: A Comparison of Manual, Semiautomated, and Fully Automated Purification Methods. Clinical Chemistry 55 (9) and in Hennig G 2010: Automated Extraction of DNA and RNA from a Single Formalin-Fixed Paraffin-Embedded Tissue Section for Analysis of Both Single-Nucleotide Polymorphisms and mRNA Expression. Clinical Chemistry 56 (12).
Bohmann et al and Hennig et al describe dissolving paraffin by heat incubation at 80° C. with simultaneous lysis of tissue sample in an aqueous solution. Following cooling of the solution to 65° C., the paraffin deposits onto the wall of the tube, and the residual tissue is removed by precipitation by means of magnetic particles (beads). Disadvantages of this method are the required incubation steps at elevated temperatures (80° C. and 65° C.) that can lead to degradation of RNA target molecules in particular. In addition, the use of magnetic beads for separation of the tissue samples leads to the risk of incomplete separation of non-lysed sample residues and potentially of the magnetic beads. This brings the risk that these components are carried-over in the subsequent purification procedure. In particular, non-lysed tissue fragments can cause clogging of pipette tips and impede further purification. With this procedure, the separation of paraffin is not controlled because of its deposition onto the wall of the tube.
Another method is known from Ribeiro-Silva A. et al 2007: RNA extraction from ten-year-old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies. BMC Molecular Biology 2 (8:118). In the method described there, the use of toxic xylene can be omitted; however, repeated washing of the sample, for example, with limonene is also required to remove the paraffin, and the sample must subsequently be rehydrated using alcohol. During removal of the wash solutions from the sample tube, the sample can again clog the pipette tip, which is also accompanied by a loss of sample material.
A further method for processing FFPE-samples is known from WO 2014/078650 A1. In this method, the paraffin is emulsified to particle sizes in the range of 10 μm by means of focused ultrasound, and the tissue components located on the opposite the side of the liquid are also removed. The use of solvents is thereby omitted. The samples are subsequently centrifuged in order to separate the emulsified paraffin from the sample. This method cannot exclude sample loss either. In addition, the method is not automatable. Potential residues of remaining paraffin can have a negative effect on binding to the purification matrix used. Emulsification of 96 samples requires an hour alone, according to the manufacturer's specifications (Covaris, truXTRAC).
In DE 10 2009 031434 A1 a method for de-paraffinization of biological samples is described in which an inert, non-hydromiscible and non-toxic solvent is used to remove the paraffin. This method can be used for single purifications. For high-throughput procedures, however, several centrifugation steps for phase separation are included that are difficult to automate.
WO2013/083260A1 describes a filter column for separating the phases of a mixture of a lysed sample from paraffin dissolved in suitable solvents, where the lysed sample and the solvent are not miscible. A conceivable disadvantage of this method is that potentially non-lysed sample components may clog the pipette tips during transfer of the lysate- and paraffin/solvent mixture to a filter column. In addition, centrifugation steps to achieve phase separation are required in this method as well. The application of a pressure gradient, for example, by means of a vacuum chamber is not described. Therefore, this method is automatable only to a limited extent due to resulting technical limitations.